A molecular replacement of AMPA receptor subunits will be used to probe the molecular basis of AMPA receptor trafficking. Long-term potentiation (LTP), a phenomenon in which brief repetitive activation of excitatory synapses results in a persistent enhancement in synaptic transmission, is the most compelling cellular model in the mammalian brain for learning and memory. Excitatory synapses release glutamate, which acts on two types of receptors;AMPA receptors (AMPARs) and NMDARs. AMPARs are hetero-tetrameric composed of combinations of the subunits GluR1-4. Evidence suggests that the change in synaptic strength during LTP is due in large part to the recruitment of AMPARs to the synapse. Two seemingly incompatible models have developed to explain AMPAR trafficking. First is the "subunit rules model" in which the C-termini of the various subunits, via their interaction with cytoplasmic scaffolding proteins, determine the mode of trafficking. Second is the "TARP-dependent trafficking model". We have discovered a family of Transmembrane AMPAR Regulatory Proteins (TARPs), which directly bind to AMPARs and to specific synaptic scaffolding proteins and are critical for surface and synaptic expression of AMPARs. The subunit rules model is entirely based on the overexpression of proteins on a wild type background, which, although powerful, has a number of limitations. In addition, these data are difficult to reconcile with conclusions from gene-targeted deletion of AMPA receptor subunits in mice. The goal of this competitive renewal is to develop a novel strategy for defining the exact role(s) of the various AMPAR subunits in trafficking. This strategy takes advantage of conditional KO technology, in which expression of Cre recombinase in single cells, excises a critical segment of the gene and acutely (~2 weeks) deletes the protein of interest. In collaboration with Dr. Peter Seeburg we will use floxed mice for GluR1, 2 and 3. Using these mice, in combination with the expression of mutated subunits in a null background, we will determine the subunit composition of synaptic and extrasynaptic AMPARs, and their role in basal and activity-dependent trafficking of receptors. These studies will provide the most definitive characterization of AMPAR trafficking and will hopefully reconcile the differences in the two prevailing models of AMPA trafficking. PUBLIC HEALTH RELEVANCE: Learning and memory is one of the most important functions of the brain and yet we know extraordinarily little about the underlying mechanisms. The elucidation of the cellular and molecular mechanisms will provide a platform for the development of a rational therapeutic approach for such diseases as Alzheimer's Disease.